Origin and arrangement of actin filaments for gliding motility in apicomplexan parasites revealed by cryo-electron tomography

The phylum Apicomplexa comprises important eukaryotic parasites that invade host tissues and cells using a unique mechanism of gliding motility. Gliding is powered by actomyosin motors that translocate host-attached surface adhesins along the parasite cell body. Actin filaments (F-actin) generated by Formin1 play a central role in this critical parasitic activity. However, their subcellular origin, path and ultrastructural arrangement are poorly understood. Here we used cryo-electron tomography to image motile Cryptosporidium parvum sporozoites and reveal the cellular architecture of F-actin at nanometer-scale resolution. We demonstrate that F-actin nucleates at the apically positioned preconoidal rings and is channeled into the pellicular space between the parasite plasma membrane and the inner membrane complex in a conoid extrusion-dependent manner. Within the pellicular space, filaments on the inner membrane complex surface appear to guide the apico-basal flux of F-actin. F-actin concordantly accumulates at the basal end of the parasite. Finally, analyzing a Formin1-depleted Toxoplasma gondii mutant pinpoints the upper preconoidal ring as the conserved nucleation hub for F-actin in Cryptosporidium and Toxoplasma. Together, we provide an ultrastructural model for the life cycle of F-actin for apicomplexan gliding motility.


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PCR are available in the EMDB under the accession codes EMD-29784, EMD-29791, and EMD-29801, respectively. Subtomogram averages of IMCSF top, sawtooth, and side views are available in the EMDB under accession codes EMD-29808, EMD-29809, and EMD-29810, respectively. The subtomogram average of the basal IMC pore is available in the EMDB under the accession code EMD-29835. Subtomogram averages of the wildtype T. gondii PCRs, refined upper PCR, and refined lower PCR are available in the EMDB under accession codes EMD-29832, EMD-29826, and EMD-29827, respectively. Subtomogram averages of the FRM1-iKD T. gondii PCRs, refined upper PCR, and refined lower PCR are available in the EMDB under the accession codes EMD-29838, EMD-29839, and EMD-29840, respectively. Source data are provided with this paper. No data was excluded from this study.
228 Cryptosporidium parvum tomograms were collected (119 untreated from three biological replicates, 109 jasplakinolide-treated from two biological replicates). 128 Toxoplasma gondii tomograms were collected (100 wildtype from three biological replicates, 28 Formin1 knockdown from one biological replicate). For each sample, except the T. gondii Formin1 knockdown, multiple frozen grids from multiple sample preparation sessions were imaged over several sessions. For the T. gondii Formin1 knockdown, one frozen grid was imaged. All attempts at replication were successful.
Samples (parasites) were allocated randomly to either be drug treated or untreated. Tomograms to be analyzed were first filtered to include those with the best contrast, and then selected at random.
No blinding efforts were taken during experiments since experimental design involved specific drug treatments and imaging that are independent of experimenter bias. No blinding efforts were taken during analyses since ground rules were set to eliminate bias. None.